Objective To validate the different expressions of human fxyd6 gene between normal bile duct tissues and malignant tumor tissues, and to observe the subcellular localization of human fxyd6 gene in human cholangiocarcinoma cells. MethodsThe different expressions between normal bile duct tissues and malignant tumor tissues were identified by RT-PCR. In situ polymerase chain reaction （IS-RT-PCR） was applied to detect the subcellular localization of fxyd6 gene in paraffin sections of human cholangiocarcinoma cells. Image analysis software was used to semiquantitatively determine the difference between normal and malignant tissues. ResultsHuman fxyd6 gene was highly expressed in cholangiocarcinoma tissues and lowly expressed in normal ones. There was a significant difference between the expressions of carcinoma cells and normal cells （P＜0.05）. IS-RT-PCR showed that fxyd6 gene localized in the kytoplasma of epithelial cells of human cholangiocarcinoma. ConclusionHuman fxyd6 gene may act as an essential component of the malignant transformation process in human cholangiocarcinoma.
Objective To construct specifically expressed vascular endothelial growth factor (VEGF)165 gene in retina. Methods Rho promoter, specifically expressed in retina, was amplified by polymerase chain reaction (PCR) from the genomic DNA of a BLAB/C rat, then it was cut with restriction enzymes and cloned into the plasmid pcDNA3.1+-VEGF165 to form recombinant plasmid pcDNA3.1+-rho-VEGF165. The correct recombinant plasmid pcDNA3.1+-rho-VEGF165 was identified by restriction enzymes and PCR, and was transferred by jetPEI into cultured human navel vein endothelial cells and human retinal pigment epithelial (RPE) cells. The expression of VEGF protein in human navel vein endothelial and RPE cells was detected by immunocytochemical staining and protraction of the growth curve of the cells. Results In human RPE cells, the expression of VEGF protein was more in recombinant plasmidpcDNA3.1+-rho-VEGF165 than that in plasmidpcDNA3.1+-rho-VEGF165 ; in human navel vein endothelial cells, no obvious difference of the expression of VEGF protein between recombinant plasmid pcDNA3.1+-rho-VEGF165 and plasmid pcDNA3.1+-rho-VEGF165 was found. Conclusions The construction of pcDNA3.1+-rho-VEGF165 carrier may provide the basic material for the study of the nosogenesis of VEGF in retinal neovascularization, and establish the foundation to set up the model of transgenic mice with VEGF specific expressing in retina. (Chin J Ocul Fundus Dis, 2005,21:106-108)
Objective To investigate whether mutations exist in codon 58 and codon 347 of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa(ADRP)． Methods Point mutations at codons 58 and 347 were detected by restriction endonuclease digestion of exons 1 and 5 amplified by polymerase chain reaction(PCR)．This method was applied to screen genomic DNAs from 57 patients of 38 families with ADRP and 60 normal controls． Results Four patients from one family of ADRP were confirmed to have a point mutation at the second nucleotide of codon 58,and 6 patients from two families of ADRP were found to have a mutation at codon 347．None of these mutations were found in 60 normal subjects． Conclusion It is suggested that molecular genetic heterogeneity exists within ADRP and some subtypes of ADRP are caused by points mutations of the rhodopsin gene． (Chin J Ocul Fundus Dis,1998,14:108-110)
PURPOSE:To investigate mitochondrial DNA(mtDNA) of Leber's hereditary optic neuropathy(LHON). METHODS:Polymerase chain reaction(PCR)method was used to analyse mtDNA of 11 patients in a pedigree with LHON and 4 control subjects from none LHON pedigree. RESULTS:There was a loss of a restriction site for the restriction endonuclease SfaN.Ⅰin Ihe Patients with LHON. In this pedigree,maternal lineage was regarded a carrier of the pathogenic gene. CONCLUSIONS:The patients with Leber's hereditary optic neuropathy have a point mutation in mtDNA,which results in loss ol SfaN I endonuclease restriction site .and this change is one of mechanisms inducing this disaese. （Chin J Ocul Fundus Dis,1997,13: 27-29）
Objective To evaluate the potential of specific mRNA marker keratin 19(K19) to detect micrometastasis by reverse transcriptase polymerase chain reaction (RT-PCR) .Methods One hundred and ninty four regional lymph nodes harvested from 6 cases of benign diseases, 4 cases of breast carcinoma, 5 cases of gastric carcinoma and 12 cases of colorectal carcinoma patients were examined by conventional pathology and amplifying tissue specific K19 mRNA by RT-PCR separately, then the two methods were compared with each other. Results None of the 34 lymph nodes which were pathological metastasis-negative from benign diseases expressed K19 mRNA by RT-PCR, all of the 28 regional lymph nodes which were pathological metastasis-positive from malignant cases showed trains of K19 mRNA by RT-PCR. Of the 132 lymph nodes which were pathological metastasis-negative from malignant cases, 11 lymph nodes were detected with micrometastasis by genetic diagnosis.Conclusion Genetic diagnosis of lymph node micrometastasis is more sensitive than conventional pathology and has diagnostic value and merits further study.
OBJECTIVE: To investigate the availability of bone defect repair by degradable porous polycaprolactam (PCL) as the carrier of bone morphogenetic protein (BMP). METHODS: Three different kinds of bone substitutes, including decalcified bone matrix, PCL-BMP compounds and simple PCL, were implanted into the radial bone defects in 36 rabbits, and in the other 12 rabbits the bone defects were left untreated as control. X-ray examination, X-ray morphometry, histological and electron microscopic observation were performed at different time after operation. RESULTS: The quantity of new bone formation in PCL-BMP group was prior to that in simple PCL group. The pattern and speed of bone defect repair in PCL-BMP group were similar to those in decalcified bone matrix. Electron microscopic observation showed that the PCL-BMP group degraded faster than simple PCL group, which was more suitable for bone repair. CONCLUSION: PCL is a good carrier of BMP with potential for clinical use.
Objective To investigate the expression of T cell receptor (TCR) Vβ8.3 gene on CD4+ T lymphocytes in the rats with experimental autoimmune uveoretinitis (EAU). Methods Eighteen Lewis rats were divided into EAU, complete Freund′s adjuvant, and the control group. Inter photoreceptor retinoid-binding protein (IRBP) R16 peptide was synthesized using Fmoc procedure for induction of EAU. Magnetic absorption cell sorting (MACS) me thod was used to isolate the CD4+T lymphocytes from the spleen of the rats. Flow cytometry was used to monitor the efficiency of isolation. The expression of TCR Vβ8.3 gene segment on CD4+T lymphocytes was determined by fluorescent quantitative polymerase chain reaction. Results EAU was successfully induced in the Lewis rats immunized with IRBP R16 peptide. The proportion of CD4+T lymphocytes isolated by means of MACS was statistically higher than that before isolation (P＜0.001). The expression of TCR Vβ8.3 gene segment on CD4+ T lymphocytes in EAU rats was significantly higher than that in the control (P＜0.05). Conclusions There is a predominant usage of antigen-specific TCR Vβ 8.3 gene in EAU rats induced by IR BP R16 peptide, which may serve as a target for immunotherapy of EAU. (Chin J Ocul Fundus Dis，2004，20：165-167)
Objective To investigate the role of β-catenin gene in breast tumorigenesis by detecting mutation and expression of β-catenin gene in breast hyperplasia and breast cancer. Methods Mutation and expression of β-catenin gene in 42 breast cancer, 15 simple hyperplasia and 15 atypical hyperplasia were detected by polymerase chain reaction-single strand conformation polymorphism and immunohistochemistry. Results Normal expression of β-catenin occurred in tissue of breast simple hyperplasia. The rate of abnormal expression of β-catenin in tissue of breast atypical hyperplasia and breast cancer were 26.7% （4/15） and 59.5% （25/42）, respectively, which were higher than that of simple hyherplasia tissue (P＜0.05). And there was a markedly difference between the atypical hyperplasia tissue and breast cancer tissue (P＜0.05). Mutation of β-catenin gene wasn’t detected in this three kinds of tissues. Conclusion Abnormal expression of β-catenin plays an important role in human breast tumorigenesis, reason of abnormal expression of β-catenin isn’t mutation of β-catenin gene. Expression of β-catenin can be regulated by other mechanisms.
Objective To observe the clinical features of acute retinal necrosis syndrome (ARN).Methods The clinical data of 84 patients (98 eyes) with ARN were retrospective analyzed. The patietns had undergone the examinations of best visual acuity, intraocular pressure, Bscanning, slitlamp biomicroscope, preset lens, direct and (or) indirect ophthalmolscope,and trihedral reflector; fundus fluorecein angiography had been performed on the patients with clear refracting media. Some of the patients had undergone polymerase chain reaction (PCR) to dectet the types of the causative virus.Medication,laser photocoagulation,and vitreous surgery had been performed on the patients after the diagnosis was confirmed. The visual acuity and the change of ocular fundus had been followed up; the average followup was 24.1 months. Results The average age of the patients at the onset was 42.8 years with the bilateraleye rate of 16.6% and retinaldetachment rate of 57.1%. There were 53.5% and 35.5% patients had the final visual acuity of gt;0.02 after 6 and 12 months, respectively. Better prognosis was found in patients diagnosed within 2 weeks and second involved eye. Varicella zoster virus DNA was identified in 15 patients and herpes simplex virus 1 was found in 3.Conclusions ARN is an acute disease with high incidence of retinal detachment.Serious retinal vasculopathy always happens at the late stage, and the prognosis is poor. Diagnosis in early stage is important and application of PCR will do contribution to the right diagnosis.
Objective To probe the association between HLA-B27 subtype and acute anterior uveitis(AAU). Methods The HLA-B27 gene of 49 AAu patients were inspected by means of polymerase chain reaction(PCR), and the B27 subtypes of AAU patients were analyzed by means of DNA sequencing and simulated nucleotide hybridization in computer. Results Twenty-nine(59.39%)of 49 AAU patients were B27 positive, and among them 13 (44.00%)were B2704 and 16(56.00%)B27052 in subtypes．No statistically significant difference in most clinical features were found between the patients with B2704 and B27052.In B27 pasitive patients,8 (seven is B27052 and one is B2704) of them were complicatde with ankylosing spondylitis(AS). Conclusions The AAU patients with either B2704 or B27052 have similar clinical feature.The B27052 gene may be an important factor for complicated AS in AAU patients. (Chin J Ocul Fundus Dis, 1999, 15: 139-142)